A major problem in the treatment of cancer and some other conditions is poor or of negligible efficiency of the specific targeting of drugs to the diseased abnormal cells and not to other unaffected cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. Many pharmaceuticals bind to cell surface receptors and reveal their action via receptor-induced processes. Pharmaceutical agents, however, often do not localize directly within the subcellular compartments of the cell which are the key sites of their action, however, meaning that the most potent action of the drug may not be achieved.
One method to target subcellular compartments that is in development attempts to target the nucleus of the cell. For example, Dinara G. Gilyazova et al. [Targeting Cancer Cells by Novel Engineered Modular Transporters, Cancer Res. 2006; 66:(21): 10534-10540], describe modular recombinant transporters that target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. The described transporters consist of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module.
U.S. Pat. No. 6,500,800 [Sobolev et al.] describes a composition for causing photodynamic damage to target cells. The composition includes a photosensitizer, a photosensitizer carrier component, a component which enables target cell recognition and transport of the photosensitizer toward the interior of the target cell by specific receptor-mediated endocytosis, and a component capable of effective targeted transport of the photosensitizer within the target cells. The composition is used to cause photodynamic damage to target cells according to the following steps: adding the composition to the cells; keeping the cells at a temperature of normal vital activity of cells with the composition for causing photodynamic damage to the target cells, the composition including the above components; and exposure of the cells to light.
U.S. Pat. No. 7,655,753 to Deonarain et al. is directed to a polypeptide comprising at least one alpha-helix having synthetically attached thereto a plurality of therapeutic or diagnostic moieties. The therapeutic or diagnostic moieties may be the same or different and are spatially oriented on the polypeptide so as to minimize interactions between the moieties. Further aspects of the '753 patent relate to a pharmaceutical composition comprising the polypeptide; a polynucleotide sequence encoding the polypeptide; an expression vector comprising said polynucleotide sequence; and a host cell transformed with said expression vector. The '753 patent also provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide.
The '753 patent appears to disclose a system that includes an alpha-helix polypeptide to which is attached a plurality of therapeutic or diagnostic moieties. The polypeptide may include two or more alpha-helical polypeptides in the form of a multi-helix bundle. The alpha-helix polypeptide of the '753 patent differs from the MTP disclosed herein in a number of respects. For example, the construct of the '753 patent appears to require assembly non-covalently from several different alpha-helix polypeptides. In contrast, the MTP disclosed herein is a single polypeptide. Further the '753 patent appears to consider only mutant forms of the ROP protein, namely the variant with four alpha-helices where at least one of them may be used for joining of an acting principle.
The '753 patent also appears to disclose the targeting protein and polypeptide being linked directly or indirectly via a linker moiety. With indirect linkage the linker moiety bonds the targeting protein to the fusion protein. Direct linkage may occur through any convenient functional group on one of the proteins, such as a hydroxy, carboxy or amino group. Indirect linkage will occur through a linking moiety. The functional groups on the linker moiety are used to form covalent bonds between the alpha helix and targeting protein.
At col. 9, lines 56-67, the '753 patent explains that the linker moiety is used for a chemical reaction via “bi- and multi-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl, aryl or aralkyl aldehydes acids esters and anyhdrides, sulphydryl or carboxyl groups, such as maleimido benzoic acid derivatives, maleimido proprionic acid derivatives and succinimido derivatives or may be derived from cyanuric bromide or chloride, carbonyldiimidazole, succinimidyl esters or sulphonic halides and the like. The functional groups on the linker moiety used to form covalent bonds between the alpha helix and targeting elements may be two or more of, e.g., amino, hydrazino, hydroxyl, thiol, maleimido, carbonyl, and carboxyl groups, etc. The linker moiety may include a short sequence of from 1 to 4 amino acid residues.” Such a linker moiety differs from the use in the instant invention of a spacer between a module and the rest of the MTP of the present invention. In one aspect of the present invention, the spacers (e.g., flexible amino-acid inserts) are used to achieve higher MTP affinity to a receptor.
The polypeptide in the '753 patent further includes a sub-cellular targeting peptide and a membrane active peptide. The sub-cellular targeting peptide may be attached either to the targeting element or to the alpha helix of the polypeptide, or to both. Examples of sub-cellular targeting peptides include nuclear localization sequences (NLS). The additional sequences can also be membrane-active peptides which function to disrupt the endosomal compartment containing the fusion protein after internalization. This will facilitate the release of the therapeutic agent into the cytosol of the cell where it can have a potent action. However, the '753 patent does not disclose a pH dependence of action of the peptides in disrupting the endosomal compartment.
As described herein, the MTP of the instant invention may include a special module which becomes membrane active only under special conditions, namely, in a slightly acidic milieau. For example, the activity of the module may have an activity maxima at pH 5.5. This action of this module is proved experimentally, described below, and has been shown to give a pore formation in lipid bilayers by the MTP of the present application under these conditions. Moreover, it should be understood that the pore formation as well as an endosomal/lysosomal activity at pH 3-pH 6 is a result of combined actions of two MTP modules, namely the endosomolytic module and the carrier module.
In one aspect, the invention described herein may have a necessity of combined action of a carrier module, HMP, and an endosomolytic module in order to make pores in lipid membranes at acidic pH's for subsequent MTP release from endosomes. It also should be added that the MTP can include a special module not only for specific subcellular targeting but also for retaining in the specific subcellular compartment of target cells (i.e., the nuclei of cancer cells).
The '753 patent also describes the therapeutic or diagnostic agent being attached directly to the polypeptide, or by virtue of a linker group. One of the properties of the MTP described herein is its ability to include an agent to be transported non-covalently, into the hydrophobic pocket of the carrier module. As described below, this can be done (i) either directly, into the porphyrin moiety that can be then inserted into the pocket, or (ii) indirectly, i.e. linked to the porphyrin derivative that can be then inserted into the pocket.
Another relevant patent is U.S. Pat. No. 6,821,948 to Braun et al, which relates to conjugates for mediating a cell-specific, compartment-specific or membrane-specific to methods of active substances. The conjugates include: a transport mediator for the cell membrane, a cell-specific, compartment-specific or membrane-specific address protein or peptide, and an active substance to be transported. In contrast to the invention described herein, the '948 patent does not disclose a module with a retention function or a non-covalent attachment of drugs.
U.S. Pat. No. 5,674,977 to Gariepy relates to a branched synthetic peptide conjugate which can be designed to bind to a target cell surface receptor, to penetrate into target cells, and to deliver a diagnostic probe or cytotoxic functionality to a desired site of action. The invention provides a relatively small molecule of flexible design having a branched structure for systematically incorporating a desired number of cytotoxic functions, peptide-based localization signals or diagnostic probes. Gariepy describes his invention as addressing problems associated with protein-based therapeutic or diagnostic agents. In contrast to the MTP of the instant invention, Gariepy's system, does not have a pH-dependent endosomolytic function; a module with a retention function; or a non-covalent attachment of drugs.
U.S. Pat. No. 6,498,233 to Wels et al. relates to a nucleic acid transfer system including a translocation domain of toxins, especially of diphtheria toxin suitable for targeting a nucleic acid, e.g., a gene, to a specific cell, and obtaining expression of the nucleic acid. The nucleic acid transfer system includes a multidomain protein component and a nucleic acid component. Wels also relates to the multidomain protein, a nucleic acid encoding the protein, suitable amplification and expression systems for the nucleic acid, and processes for their preparation and uses. In contrast to the invention described herein, Wels does not disclose a compartment-specific function, a retention function, or a function for transport of nucleic acids. In one aspect of the inventions described herein, the MTPs of the instant application may have an endosomolytic module representing a truncated diphtheria toxin translocation domain. This truncation (202-384 aa) was made to discard the cleavable protease site after 194 amino acid as well as 186 and 201 Cysteins, which subtend the cleavable amino acid loop.
U.S. Pat. No. 5,965,406 to Murphy is directed to a recombinant DNA molecule encoding a hybrid protein comprising a first part, a second part, and a third part. The first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal. The second part comprises a portion of a translocation domain of naturally occurring protein selected from the group consisting of diphtheria toxin, botulinum neurotoxin, ricin, cholera toxin, LT toxin, C3 toxin, Shiga toxin, Shiga-like toxin, pertussis toxin and tetanus toxin, which translocates said third part across the cytoplasmic membrane into the cytosol of the cell. The third part comprises a polypeptide entity to be introduced into the cell. The third part is non-native with respect to the naturally occurring protein of the second part.
In contrast to the MTP described herein, the '406 patent does not disclose a compartment-specific function, a retention function, or the non-covalent attachment of drugs. Further, the MTPs described herein generally will have at least four modules necessary for targeted intranuclear delivery and, are not restricted to only a concrete translocation domain or a domain from the toxin group. In addition, as described above, the MTPs of the instant application may have an endosomolytic module representing a truncated diphtheria toxin translocation domain.
U.S. Pat. No. 6,022,950 to Murphy, similarly discloses a hybrid molecule comprising a first part, a second part, and a third part connected by covalent bonds. The first part includes a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal. The second part comprises a portion of a translocation domain of naturally occurring protein which translocates said third part across the cytoplasmic membrane into the cytosol of the cell. The third part comprises a chemical entity to be introduced into the cell. The first part and third part are non-native with respect to the naturally occurring protein, and further the covalent bond connecting the second part and the third part is a cleavable bond. When the second part comprises a portion of a translocation domain of Pseudomonas exotoxin, the third part is not a polypeptide. The description notes that MSH can selectively bind to melanocytes, rendering hybrids, once labelled with a detectable label, useful in the diagnosis of melanoma and the in vivo and in vitro detection of metastic melanoma loci. Such a hybrid, when attached to an enzymatically-active portion of a toxin molecule instead of to a detectable label, could be utilized to deliver that toxic activity specifically to the target melanoma cells
In contrast to the MTP described herein, the '950 patent does not disclose a retention function, the non-covalent attachment of drugs or any modules/functions for translocation across the cytoplasmic membrane into the cytosol of the cell.